EP2644619A1 - Domaine N-terminal monomère de la protéine de soie d'araignée et ses utilisations - Google Patents

Domaine N-terminal monomère de la protéine de soie d'araignée et ses utilisations Download PDF

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EP2644619A1
EP2644619A1 EP12162642.8A EP12162642A EP2644619A1 EP 2644619 A1 EP2644619 A1 EP 2644619A1 EP 12162642 A EP12162642 A EP 12162642A EP 2644619 A1 EP2644619 A1 EP 2644619A1
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protein
fusion protein
polypeptide
moiety
proteins
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English (en)
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Jan Johansson
Kerstin Norling
Anna Rising
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Spiber Technologies AB
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Spiber Technologies AB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

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  • the present invention relates to the field of proteins and polypeptides, and more specifically to expression and production of spider silk proteins (spidroins) and other, non-spidroin proteins and polypeptides.
  • the present invention provides novel proteins which are useful in themselves and as a moiety in novel fusion proteins for expression and production of the desired proteins and polypeptides, as well as nucleic acid molecules encoding these novel proteins and fusion proteins.
  • the present invention also provides a method of expressing and producing a desired protein or polypeptide.
  • Production of proteins and polypeptides from DNA can be achieved in various hosts, but a common problem is the formation of insoluble protein/polypeptide aggregates. This may severely impede or even prevent production of a functional protein/polypeptide.
  • One solution to this problem is to express the desired protein or polypeptide as a fusion protein with a protein or polypeptide that provides the required solubility.
  • the fusion protein may be cleaved, and the desired protein isolated.
  • the desired protein/polypeptide may be maintained integrated in the soluble fusion protein, where it remains functional and can be subjected to further characterization, e.g. activity studies, structure determination and crystallization.
  • SP C lung surfactant protein C
  • SP-C33 is a variant of SP-C, where the residues in the transmembrane helix (normally mainly valines) are exchanged for leucines.
  • SP-C33 retains the function of native SP-C, including proper insertion in membranes, but is less prone to aggregate and therefore feasible to produce in large quantities for development of a synthetic surfactant preparation. Since SP-C33 so far has not been possible to produce from DNA, it is today manufactured by chemical synthesis.
  • proteins and polypeptides that pose difficulties when expressed from recombinant DNA are A ⁇ -peptide, IAPP, PrP, ⁇ -synuclein, calcitonin, prolactin, cystatin, ATF and actin; SP-B, ⁇ -defensins and ⁇ -defensins; class A-H apolipoproteins; LL-37, hCAP18, SP-C, SP-C33Leu, Brichos, GFP, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; avidin and streptavidin; and protease 3C.
  • WO 2011/115538 discloses a fusion protein comprising a solubility-enhancing moiety which is derived from the N-terminal (NT) fragment of a spider silk protein and a moiety which is a desired protein or polypeptide.
  • NT N-terminal
  • a pH above 6.4 is preferred to prevent assembly of the solubility-enhancing moiety.
  • NT N-terminal
  • the present invention provides compositions comprising aqueous solutions of said proteins as set out in the appended claims.
  • the present invention provides isolated nucleic acids encoding the proteins as set out in the appended claims.
  • the present invention provides useful applications of a novel protein as such and as a moiety in a fusion protein.
  • the present invention provides a novel method of producing a desired protein or polypeptide as set out in the appended claims.
  • the present invention provides a novel method of producing a fusion protein comprising a desired protein or polypeptide as set out in the appended claims.
  • the present invention is concerned with production and expression of proteins and polypeptides.
  • the end product may vary. It may for instance be desirable to obtain the protein or polypeptide inserted in a lipid membrane, in solution or associated with other biomolecules. It shall also be realized that it may also be highly desirable to obtain the desired protein or polypeptide as part of a fusion protein, which may provide a suitable handle for purification and detection and/or provide desirable properties, e.g. stability and solubility. Maintaining the desired protein or polypeptide functionally integrated in a soluble fusion protein is useful to characterize and study the desired protein or polypeptide.
  • the present invention is generally based on the insight of the usefulness of a specific variant of the N-terminal (NT) fragment of a spider silk protein due to its capacity to be present as a soluble monomer regardless of the pH of the surrounding aqueous medium.
  • the present invention provides according to a first aspect a protein comprising a moiety of 100-160 amino acid residues having at least 80% identity with SEQ ID NO 1, wherein the amino acid residue corresponding to position 72 in SEQ ID NO 1 is not Ala or Gly. While this particular amino acid residue is alanine or glycine in known spider silk protein species, it is shown in the Examples that mutation of this particular residue has a critical impact on the pH-dependent dimerization capacity of the NT fragment.
  • Wildtype NT is highly water-soluble and useful e.g. as a solubility-increasing moiety in a fusion protein for the expression of a desired protein or polypeptide, but it also form dimers at a pH interval of 4.2-6.3 which increases the risk of undesirable aggregation of the fusion proteins.
  • This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide. It is also a useful pH interval for certain purification protocols, e.g. when using cation exchange as a purification principle.
  • mutant NT protein according to the invention is therefore useful in itself to study the physiologically relevant NT monomer as such.
  • the mutant NT protein according to the invention is also useful as a solubility-increasing moiety in a fusion protein, since it decreases the risk of undesirable aggregation of the fusion proteins, and thereby opens up a new pH window (4.2-6.3) in which derivatives of wildtype NT from spider silk protein can be used in biochemical applications when solubility of protein/polypeptide monomers in aqueous solutions is desirable, e.g. in production or characterization of desirable proteins or polypeptides.
  • the position corresponding to position 72 of wildtype NT is a charged and/or bulky amino acid selected from the group consisting of Arg, Lys, His, Glu, Asp, Gln, Asn, Tyr, Thr and Ser.
  • it is preferred that in the position corresponding to position 72 of wildtype NT is a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp, especially the positively charged amino acids Arg, Lys and His.
  • the position corresponding to position 72 of wildtype NT is arginine.
  • the inventive moiety is derived from the NT fragment of a spider silk protein, or spidroin.
  • spidroins proteins derived from major spidroin 1 (MaSp1) from Euprosthenops australis
  • MaSp1 major spidroin 1
  • spike silk proteins are used interchangeably throughout the description and encompass all known spider silk proteins, including major ampullate spider silk proteins which typically are abbreviated "MaSp", or "ADF” in the case of Araneus diadematus.
  • MaSp major ampullate spider silk proteins
  • ADF Araneus diadematus.
  • These major ampullate spider silk proteins are generally of two types, 1 and 2. These terms furthermore include the new NT protein fragments according to the invention, as defined in the appended claims and itemized embodiments, and other non-natural proteins with a high degree of identity and/or similarity to the known spider silk NT protein fragments.
  • the inventive moiety has a high degree of similarity to the N-terminal (NT) amino acid sequence of spider silk proteins. As shown in Fig 1 , this amino acid sequence is well conserved among various species and spider silk proteins, including MaSp1 and MaSp2. The skilled person is therefore well aware how, and to what extent, the amino acid sequence may be varied without departing from the properties and functionality of the N-terminal spider silk protein fragment.
  • Fig 1 the following spidroin NT fragments are aligned, denoted with GenBank accession entries where applicable: TABLE 1 - Spidroin NT fragments Code Species and spidroin protein GenBank acc. no.
  • Nc flag and Nlm flag are translated and edited according to Rising A. et al. Biomacromolecules 7, 3120-3124 (2006 )).
  • the NT moiety according to the invention can be selected from any of the amino acid sequences shown in Fig 1 or sequences with a high degree of similarity. A wide variety of sequences can be used in the fusion protein according to the invention.
  • Fig 1 Based on the homologous sequences of Fig 1 , the following sequence constitutes a consensus NT amino acid sequence: QANTPWSSPNLADAFINSF(M/L)SA(A/I)SSSGAFSADQLDDMSTIG(D/N/Q)T LMSAMD(N/S/K)MGRSG(K/R)STKSKLQALNMAFASSMAEIAAAESGG(G/Q) SVGVKTNAISDALSSAFYQTTGSVNPQFV(N/S)EIRSLI(G/N)M(F/L)(A/S)QAS ANEV (SEQ ID NO 4).
  • sequence of the inventive moiety according to the invention has at least 50% identity, preferably at least 60% identity, to the consensus amino acid sequence SEQ ID NO 4, which is based on the wildtype NT amino acid sequences of Fig 1 .
  • sequence of the inventive moiety according to the invention has at least 65% identity, preferably at least 70% identity, to the consensus amino acid sequence SEQ ID NO 4.
  • solubility-enhancing moiety according to the invention has furthermore 70%, preferably 80%, similarity to the consensus amino acid sequence SEQ ID NO 4.
  • a representative inventive moiety according to the invention is SEQ ID NO 1 (encoded by SEQ ID NO 2), which is derived from the Euprosthenops australis NT moiety SEQ ID NO 3 with replacement of alanine in position 72 as set out hereinabove.
  • the inventive moiety has at least 80% identity to SEQ ID NO 1 or any individual amino acid sequence in Fig 1 .
  • the inventive moiety has at least 90%, such as at least 95% identity, to SEQ ID NO 1 or any individual amino acid sequence in Fig 1 .
  • the solubility-enhancing moiety is identical to SEQ ID NO 1 or any individual amino acid sequence in Fig 1 , with the proviso that alanine in position 72 is replaced as set out hereinabove.
  • % identity is calculated as follows.
  • the query sequence is aligned to the target sequence using the CLUSTAL W algorithm ( Thompson, J.D., Higgins, D.G. and Gibson, T.J., Nucleic Acids Research, 22: 4673-4680 (1994 )).
  • a comparison is made over the window corresponding to the shortest of the aligned sequences.
  • the amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % identity.
  • % similarity is calculated as described for "% identity", with the exception that the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys are similar; the basic residues Lys, Arg and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gln, Asn, Ser, Thr and Tyr are similar.
  • the remaining natural amino acid Gly is not similar to any other amino acid in this context.
  • alternative embodiments according to the invention fulfill, instead of the specified percentage of identity, the corresponding percentage of similarity.
  • Other alternative embodiments fulfill the specified percentage of identity as well as another, higher percentage of similarity, selected from the group of preferred percentages of identity for each sequence.
  • a sequence may be 70% similar to another sequence; or it may be 70% identical to another sequence; or it may be 70% identical and 90% similar to another sequence.
  • the inventive moiety contains from 100 to 160 amino acid residues. It is preferred that the inventive moiety contains at least 100, or more than 110, preferably more than 120, amino acid residues. It is also preferred that the inventive moiety contains at most 160, or less than 140 amino acid residues. A typical inventive moiety contains approximately 130-140 amino acid residues.
  • the N-terminal (NT) fragment of a spider silk protein is particularly useful as a solubility-enhancing moiety in a fusion protein that is produced from recombinant DNA.
  • the present invention is further based on the insight of the usefulness of a specific variant of the N-terminal (NT) fragment of a spider silk protein in such a fusion protein due to its capacity to be present as a soluble monomer regardless of the pH of the surrounding aqueous medium.
  • the present invention provides a fusion protein comprising (i) at least one solubility-enhancing moiety of 100-160 amino acid residues having at least 80% identity with SEQ ID NO 1, wherein the amino acid residue corresponding to position 72 in SEQ ID NO 1 is not Ala or Gly; and (ii) at least one moiety which is a desired protein or polypeptide.
  • Preferred features of the solubility-enhancing moiety are presented hereinabove. Is has surprisingly been determined from experimental data that although this change in position 72 decreases the flexibility of the NT structure and thereby locks the protein into the monomeric state, its capacity to provide stability and solubility to the desirable protein/polypeptide moiety in the fusion protein according to the invention is maintained.
  • the fusion proteins consists of (i) at least one solubility-enhancing moiety of 100-160 amino acid residues having at least 80% identity with SEQ ID NO 1, wherein the amino acid residue corresponding to position 72 in SEQ ID NO 1 is not Ala or Gly; and (ii) at least one moiety which is a desired protein or polypeptide, optionally including other preferred features disclosed herein, e.g. a linker peptide and/or a cleavage site between the solubility-enhancing moiety and the desired protein or polypeptide.
  • a linker peptide and/or a cleavage site between the solubility-enhancing moiety and the desired protein or polypeptide e.g. a linker peptide and/or a cleavage site between the solubility-enhancing moiety and the desired protein or polypeptide.
  • the fusion protein may be useful as such in isolated form, e.g. for studies of otherwise aggregated or poorly soluble proteins in soluble form, or in crystallization associated
  • fusion protein implies here a protein that is made by expression from a recombinant nucleic acid, i.e. DNA or RNA that is created artificially by combining two or more nucleic acid sequences that would not normally occur together (genetic engineering).
  • the fusion proteins according to the invention are recombinant proteins, and they are therefore not identical to naturally occurring proteins.
  • the combined nucleic acid sequences encode different proteins, partial proteins or polypeptides with certain functional properties.
  • the resulting fusion protein, or recombinant fusion protein is a single protein with functional properties derived from each of the original proteins, partial proteins or polypeptides.
  • the fusion protein according to the invention and the corresponding genes are chimeric, i.e. the protein/gene fragments are derived from at least two different species.
  • the solubility-enhancing moiety is derived from the N-terminal fragment of a spider silk protein.
  • the desired protein or polypeptide is a non-spidroin protein. This implies that the desired protein or polypeptide is not derived from the C-terminal, repetitive or N-terminal fragment of a spider silk protein.
  • the fusion protein according to the invention may also contain one or more linker peptides.
  • the linker peptide(s) may be arranged between the solubility-enhancing moiety and the desired protein or polypeptide moiety, or may be arranged at either end of the solubility-enhancing moiety and the desired protein or polypeptide moiety. If the fusion protein contains two or more solubility-enhancing moieties, the linker peptide(s) may also be arranged in between two solubility-enhancing moieties.
  • the linker(s) may provide a spacer between the functional units of the fusion protein, but may also constitute a handle for identification and purification of the fusion protein, e.g. a His and/or a Trx tag.
  • the fusion protein contains two or more linker peptides for identification and purification of the fusion protein, it is preferred that they are separated by a spacer sequence, e.g. His 6 -spacer-His 6 -.
  • the linker may also constitute a signal peptide, such as a signal recognition particle substrate, which directs the fusion protein to the membrane and/or causes secretion of the fusion protein from the host cell into the surrounding medium.
  • the fusion protein may also include a cleavage site in its amino acid sequence, which allows for cleavage and removal of the linker(s) and/or the solubility-enhancing moiety or moieties.
  • cleavage sites are known to the person skilled in the art, e.g.
  • cleavage sites for chemical agents such as CNBr after Met residues and hydroxylamine between Asn-Gly residues
  • cleavage sites for proteases such as thrombin or protease 3C
  • self-splicing sequences such as intein self-splicing sequences.
  • the desirable protein/polypeptide is expressed as a C-terminal fusion to His-protease 3C cleavage site-NT A72R -TEV protease cleavage site, see. e.g. Example 5.
  • Each solubility-enhancing moiety is linked, directly or indirectly, to the desired protein or polypeptide moiety.
  • a direct linkage implies a direct covalent binding between the two moieties without intervening sequences, such as linkers.
  • An indirect linkage also implies that the two moieties are linked by covalent bonds, but that there are intervening sequences, such as linkers and/or one or more further solubility-enhancing moieties.
  • the at least one solubility-enhancing moiety may be arranged at either end of the desired protein or polypeptide, i.e. C-terminally arranged or N-terminally arranged. It is preferred that the least one solubility-enhancing moiety is arranged at the N-terminal end of the desired protein or polypeptide. If the fusion protein contains one or more linker peptide(s) for identification and purification of the fusion protein, e.g. a His or Trx tag(s), it is preferred that it is arranged at the N-terminal end of the fusion protein.
  • the at least one solubility-enhancing moiety may also be integrated within the desired protein or polypeptide, for instance between domains or parts of a desired protein.
  • At least one solubility-enhancing moiety constitutes the N-terminal and/or the C-terminal end of the fusion protein, i.e. no linker peptide or other sequence is present terminal of the solubility-enhancing moiety.
  • a typical fusion protein according to the invention may contain 1-6, such as 1-4, such as 1-2 solubility-enhancing moieties.
  • the fusion protein is comprising at least two solubility-enhancing moieties, each being derived from the N-terminal (NT) fragment of a spider silk protein as set out hereinabove.
  • the solubility-enhancing moieties preferably two solubility-enhancing moieties, may be consecutively arranged at either end of the desired protein or polypeptide, i.e. C-terminally arranged or N-terminally arranged. Consecutively arranged solubility-enhancing moieties may also be integrated within the desired protein or polypeptide, for instance between domains or parts of a desired protein.
  • the solubility-enhancing moieties may also be non-consecutively arranged, either at each end of the desired protein or polypeptide, i.e. C-terminally and N-terminally arranged, or at one end of the desired protein or polypeptide and integrated within the desired protein or polypeptide.
  • a typical preferred fusion protein according to the invention may contain 2-6, such as 2-4 solubility-enhancing moieties.
  • the fusion protein according to the invention has at least one cleavage site arranged between at least one desired protein or polypeptide moiety and at least one solubility-enhancing moiety. This allows for cleavage of the fusion protein and purification of the desired protein. It is however noted that it may be desirable to obtain the desired protein or polypeptide as part of a fusion protein, which may provide a suitable handle for purification and detection and/or provide desirable properties, e.g. stability and solubility. In this case, the cleavage site may be omitted, or the cleavage site may be included but the cleavage step omitted.
  • a preferred fusion protein has the form of an N-terminally arranged solubility-enhancing moiety, coupled by a linker peptide of 1-30 amino acid residues, such as 1-10 amino acid residues, to a C-terminally arranged desired protein or polypeptide.
  • the linker peptide may contain a cleavage site.
  • the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.
  • Another preferred fusion protein has the form of an N-terminally arranged solubility-enhancing moiety coupled directly to a C-terminally arranged desired protein or polypeptide.
  • the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.
  • One preferred fusion protein has the form of a two consecutive N-terminally arranged solubility-enhancing moieties, coupled by a linker peptide of 1-30 amino acid residues, such as 1-10 amino acid residues, to a C-terminally arranged desired protein or polypeptide.
  • the linker peptide may contain a cleavage site.
  • the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.
  • Another preferred fusion protein has the form of two consecutive N-terminally arranged solubility-enhancing moieties coupled directly to a C-terminally arranged desired protein or polypeptide.
  • the fusion protein has an N-terminal or C-terminal linker peptide, which may contain a purification tag, such as a His tag, and a cleavage site.
  • the desired protein or polypeptide is selected from the group consisting of amyloid-forming proteins and polypeptides, disulphide-containing proteins and polypeptides, apolipoproteins, membrane proteins and polypeptides, protein and polypeptide drugs and drug targets, aggregation-prone proteins and polypeptides, and proteases.
  • a preferred group of desired proteins or polypeptides is consisting of A ⁇ -peptide, IAPP, PrP, ⁇ -synuclein, calcitonin, prolactin, cystatin, ATF and actin; SP-B, mini-BLeu, ⁇ -defensins and ⁇ -defensins; class A-H apolipoproteins; LL-37, hCAP18, SP-C, SP-C33, SP-C33Leu, Brichos, GFP, neuroserpin; hormones, including EPO and GH, and growth factors, including IGF-I and IGF-II; avidin and streptavidin; and protease 3C.
  • Amyloid-forming proteins and polypeptides according to the invention include proteins and polypeptides that are associated with disease and functional amyloid.
  • amyloid-forming proteins and polypeptides include amyloid beta peptide (A ⁇ -peptide), islet amyloid polypeptide (amylin or IAPP), prion protein (PrP), ⁇ -synuclein, calcitonin, prolactin, cystatin, atrial natriuretic factor (ATF) and actin.
  • a ⁇ -peptide amyloid beta peptide
  • IAPP islet amyloid polypeptide
  • PrP prion protein
  • ⁇ -synuclein calcitonin
  • prolactin prolactin
  • cystatin cystatin
  • atrial natriuretic factor ATF
  • actin actin. Examples of amyloid-forming proteins and polypeptides according to the invention are listed in Table 2.
  • solubility-enhancing moiety promotes the desired formation of intrachain disulphide bonds over interchain disulphide bonds in defensins and other disulphide-containing proteins and polypeptides.
  • disulphide-containing proteins and polypeptides according to the invention are listed in Table 3.
  • apolipoproteins examples are listed in Table 4. TABLE 4 - Apolipoproteins Protein Sequence / Uniprot ID Apolipoprotein B-100 P04114 Apolipoprotein C-1 P02654 Apolipoprotein D P05090 Apolipoprotein E P02649
  • membrane proteins and polypeptides include membrane-associated receptors, including cytokine receptors, KL4, LL-37, hCAP18, surfactant protein C (SP-C) and variants thereof, such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu.
  • SP-C surfactant protein C
  • Other specific examples include SP-C33Leu fused to Mini-B,Mini-BLeu, 1 a AA, 1 b AA, 0 AAAA, 1 a LL, 1 b LL, 0 LLLL or SP-B proteins, optionally via a linker, e.g. Gly n , Leu n , Gly-Ala n or the like.
  • SP-C33Leu may be arranged N-terminal or, preferably, C-terminal to the Mini-B,Mini-BLeu, 1a AA, 1b AA, 0 AAAA, 1a LL, 1 b LL, 0 LLLL or SP-B protein.
  • Mini-B,Mini-BLeu 1a AA, 1b AA, 0 AAAA, 1a LL, 1 b LL, 0 LLLL or SP-B protein.
  • membrane proteins and polypeptides according to the invention are listed in Table 5.
  • protein and polypeptide drugs and drug targets include hormones that are produced recombinantly, including peptide and protein hormones, such as erythropoietin (EPO) and growth hormone (GH), cytokines, growth factors, such as insulin-like growth factors (IGF-I and IGF-II), KL4, LL-37, hCAP18, surfactant protein C (SP-C) and variants thereof, such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu.
  • EPO erythropoietin
  • GH growth hormone
  • cytokines growth factors
  • growth factors such as insulin-like growth factors (IGF-I and IGF-II)
  • KL4, LL-37 LL-37
  • hCAP18 surfactant protein C
  • SP-C surfactant protein C
  • variants thereof such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu.
  • SP-C33Leu fused to Mini-B,Mini-BLeu, 1 a AA, 1 b AA, 0 AAAA, 1 a LL, 1 b LL, 0 LLLL or SP-B proteins, optionally via a linker, e.g. Gly n , Leu n , Gly-Ala n or the like.
  • SP-C33Leu may be arranged N-terminal or, preferably, C-terminal to the Mini-B,Mini-BLeu, 1 a AA, 1 b AA, 0 AAAA, 1 a LL, 1b LL, 0 LLLL or SP-B protein.
  • Protein and polypeptide drugs and drug targets according to the invention are listed in Table 6. TABLE 6 - Protein and polypeptide drugs and drug targets Protein Sequence / Uniprot ID Insulin-like growth factor IA P01243 Insulin like growth factor IB P05019 Growth hormone 1, variant 1 Q61YF1 Growth hormone 1, variant 2 Q61YF0 Growth hormone 2, variant 2 B1A4H7 Insulin P01308 Erythropoietin P01588 Coagulation Factor VIII P00451 Coagulation Factor IX P00740 Prothrombin P00734 Serum albumin P02768 Antithrombin III P01008 Interferon alfa P01563 Somatotropin P01241 Major pollen allergen Bet v 1-A P15494 OspA (Piscirickettsia salmonis) Q5BMB7 17 kDa antigen variant of OspA ( P.
  • aggregation-prone proteins and polypeptides examples include avidin, streptavidin and extracellular, ligand-binding parts of cytokine receptors. Examples of aggregation-prone proteins and polypeptides according to the invention are listed in Table 7.
  • proteases examples include protease 3C from coxsackie virus or human rhinovirus. Further examples of proteases according to the invention are listed in Table 8. TABLE 8 - Proteases Protease Class Accession no. Trypsin (bovine) serine P00760 Chymotrypsin (bovine) serine P00766 Elastase (porcine) serine P00772 Endoproteinase Arg-C (mouse submaxillary gland) serine Endoproteinase Glu-C (V8 protease) (Staphylococcus aureus) serine P04188 Acylamino-acid-releasing enzyme (porcine) serine P19205 Carboxypeptidase (Penicillium janthinellum) serine P43946 Proteinase K (Tritirachium album) serine P06873 Subtilisin (Bacillus subtilis) serine P04189 P29122 Carboxypeptidase Y (
  • the desired protein or polypeptide is selected from surfactant protein B (SP-B) and variants thereof, such as Mini-B, Mini-B27, Mini-BLeu, KL4, LL-37, hCAP18, and surfactant protein C (SP-C) and variants thereof, such as SP-C(Leu), SP-C33, SP-C30 and SP-C33Leu.
  • SP-B surfactant protein B
  • SP-C surfactant protein C
  • Other preferred non-spidroin proteins according to the invention are neuroserpin, GFP, and the 1 a AA, 1 b AA, 0 AAAA, 1 a LL, 1 b LL and 0 LLLL proteins.
  • the fusion protein is selected from the group consisting of SEQ ID NOS 6, 8, 10, and 12-34; and proteins having at least 80%, preferably at least 90%, more preferably at least 95% identity, to any of these proteins.
  • the present invention provides a composition comprising an aqueous solution of a protein according to the invention.
  • the composition is consisting of an aqueous solution of a protein according to the invention.
  • the protein is a fusion protein according to the invention.
  • the pH of the composition is 6.3 or lower, such as 4.2-6.3.
  • the present invention provides an isolated nucleic acid encoding a protein according to the invention.
  • the isolated nucleic acid is selected from the group consisting of SEQ ID NOS 2, 5, 7, 9 and 11.
  • the present invention provides a novel use of at least one moiety of 100-160 amino acid residues having at least 80% identity with SEQ ID NO 1, wherein the amino acid residue corresponding to position 72 in SEQ ID NO 1 is not Ala or Gly, as a moiety in a fusion protein for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide.
  • Preferred features of the inventive moiety are presented hereinabove.
  • the solubility-enhancing moiety is used for production of the desired protein or polypeptide. In another preferred embodiment, the solubility-enhancing moiety is used for studying or characterizing the desired protein or polypeptide.
  • an advantageous use of the inventive moiety is as a solubility-enhancing moiety in a fusion protein which is subjected to a pH of 6.3 or lower, such as 4.2-6.3.
  • a pH of 6.3 or lower such as 4.2-6.3.
  • This specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wildtype NT forms dimers at a pH interval of 4.2-6.3 which increases the risk of undesirable aggregation of the fusion proteins.
  • This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide. It is also a useful pH interval for certain purification protocols, e.g. when using cation exchange as a purification principle.
  • the present invention provides a method of producing a fusion protein.
  • the first step involves expressing in a suitable host a fusion protein according to the invention.
  • Suitable hosts are well known to a person skilled in the art and include e.g. bacteria and eukaryotic cells, such as yeast, insect cell lines and mammalian cell lines.
  • this step involves expression of a nucleic acid molecule which encodes the fusion protein in E. coli.
  • the second method step involves obtaining a mixture containing the fusion protein.
  • the mixture may for instance be obtained by lysing or mechanically disrupting the host cells.
  • the mixture may also be obtained by collecting the cell culture medium, if the fusion protein is secreted by the host cell.
  • the thus obtained protein can be isolated using standard procedures. If desired, this mixture can be subjected to centrifugation, and the appropriate fraction (precipitate or supernatant) be collected.
  • the mixture containing the fusion protein can also be subjected to gel filtration, chromatography, e.g. cation exchange chromatography, dialysis, phase separation or filtration to cause separation.
  • lipopolysaccharides and other pyrogens are actively removed at this stage.
  • the obtained mixture comprises the fusion protein dissolved in a liquid medium, typically a salt buffer or cell culture medium.
  • a liquid medium typically a salt buffer or cell culture medium.
  • the mixture has a pH of 6.3 or lower, such as 4.2-6.3.
  • the present invention provides a method of producing a desired protein or polypeptide.
  • the first step involves expressing in a suitable host a fusion protein according to the invention.
  • Suitable hosts are well known to a person skilled in the art and include e.g. bacteria and eukaryotic cells, such as yeast, insect cell lines and mammalian cell lines.
  • this step involves expression of a nucleic acid molecule which encodes the fusion protein in E. coli.
  • the second method step involves obtaining a mixture containing the fusion protein.
  • the mixture may for instance be obtained by lysing or mechanically disrupting, e.g. sonicating, the host cells.
  • the mixture may also be obtained by collecting the cell culture medium, if the fusion protein is secreted by the host cell.
  • the thus obtained protein can be isolated using standard procedures. If desired, this mixture can be subjected to centrifugation, and the appropriate fraction (precipitate or supernatant) be collected.
  • the mixture containing the fusion protein can also be subjected to gel filtration, chromatography, e.g. cation exchange chromatography, dialysis, phase separation or filtration to cause separation.
  • linker peptides may be removed by cleavage in this step. As set out above, this may be the most suitable form of the desired protein or polypeptide, i.e. as part of a fusion protein. It may provide a suitable handle for purification and detection and/or provide desirable properties, e.g. stability and in particular solubility.
  • the method may also comprise the step of cleaving the fusion protein to provide the desired protein or polypeptide.
  • the fusion protein is comprising at least one cleavage site arranged between at least one desired protein or polypeptide moiety and at least one solubility-enhancing moiety. In a typical fusion protein, this implies the presence of a single cleavage site between the solubility-enhancing moiety or moieties and the desired protein or polypeptide.
  • Cleavage may be achieved using standard procedures, for instance cleavage by cyanogen bromide (CNBr) after Met residues, cleavage by hydroxylamine between Asn and Gly residues, cleavage by protease 3C between Gln and Gly residues at -XLETLFQGX- sites, and at various other protease sites that are well known to the person skilled in the art.
  • CBr cyanogen bromide
  • the thus obtained desired protein or polypeptide can be isolated using standard procedures. If desired, this mixture can be subjected to centrifugation, and the appropriate fraction (precipitate or supernatant) be collected. The mixture containing the desired protein or polypeptide can also be subjected to gel filtration, chromatography, dialysis, phase separation or filtration to cause separation. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage. If desired, linker peptides may be removed by cleavage in this step.
  • the obtained mixture comprises the fusion protein dissolved in a liquid medium, typically a salt buffer or cell culture medium.
  • the mixture has a pH of 6.3 or lower, such as 4.2-6.3.
  • This specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium. Wildtype NT forms dimers at a pH interval of 4.2-6.3 which increases the risk of undesirable aggregation of the fusion proteins.
  • This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide, e.g. amyloid-forming or aggregation-prone proteins/polypeptides. It is also a useful pH interval for certain purification protocols, e.g. when using cation exchange as a purification principle.
  • step b) further involves purification of the fusion protein on a cation exchange medium.
  • the fusion protein is typically obtained as a solution in a liquid medium.
  • soluble and “in solution” is meant that the fusion protein is not visibly aggregated and does not precipitate from the solvent at 60 000 ⁇ g.
  • the liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as a 10-50 mM Tris-HCl buffer or phosphate buffer.
  • solubility-enhancing moiety improves the stability of the desired protein/polypeptide and prevents moiety dimer formation under these conditions.
  • This can be advantageous when immediate polymerisation may be undesirable, e.g. during protein purification or in preparation of large batches.
  • methods according to the invention which are comprising at least one step involves subjecting the fusion protein to a pH of 6.3 or lower, such as 4.2-6.3.
  • this specific variant of the N-terminal (NT) fragment of a spider silk protein is present as a soluble monomer regardless of the pH of the surrounding aqueous medium.
  • Wildtype NT forms dimers at a pH interval of 4.2-6.3 which increases the risk of undesirable aggregation of the fusion proteins. This is a useful pH interval for the functionality and stability of certain desirable proteins and polypeptide, e.g. amyloid-forming or aggregation-prone proteins/polypeptides.
  • An expression vector was constructed to produce NT as a C-terminal fusion to His 6 -thioredoxin-His 6 .
  • the mutation A72R (SEQ ID NO 5) was introduced in the NT gene, and the mutant (SEQ ID NO 6) and wt NT were expressed and purified.
  • E. coli BL21 (DE3) cells were grown at 37°C in the presence of kanamycin, either in minimal medium M9 containing 15 N-ammonium acetate and 13 C-glucose (for NMR studies), or in LB medium (for all other analyses). Protein expression was induced by addition of isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), and the cells were thereafter kept for 4 h at 30°C.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • NT SEQ ID NO 3
  • NT A72R SEQ ID NO 1
  • Fluorescence emission spectra between 300 and 400 nm were recorded for the proteins NT and NT A72R at 5 ⁇ M concentration in 10 mM Hepes/Mes, pH values between 5.6 and 7.6, using a Fluorolog-3 instrument, excitation wavelength 280 nm, emission and excitation slit widths 5 nm.
  • NT A72R solid line
  • wild type NT wild type NT
  • NT A72R 1 mg/mL was incubated at 22°C in 90% D 2 O in 18 mM Tris-HCl at pH 6.0 or 7.0. Incubations were started by diluting a 10 mg/mL protein solution 1:10 in buffer prepared in 99.9% D 2 O (Cambridge Isotopes, Andover, MA). Aliquots were collected from 1 min and onwards, quenched by addition of trifluoroacetic acid (TFA) (Merck, Darmstadt, Germany), and frozen in liquid nitrogen. Samples were kept in liquid nitrogen until analyzed. For MS, aliquots of deuterated NT A72R were thawed and injected into an HPLC system submersed in an ice bath.
  • TFA trifluoroacetic acid
  • Protein samples were digested online in a Porozyme Immobilized Pepsin Cartridge (Applied Biosystems, Foster City, CA) operated at 17 ⁇ L/min in 0.05% TFA. Peptic peptides were desalted using a Waters Symmetry C 18 trap column and eluted in a single step with 70% acetonitrile containing 0.1 % formic acid at a flow rate of 17 ⁇ L/min.
  • Samples were delivered to the mass spectrometer through a tapered tip emitter with an opening of 50 ⁇ m (New Objective, Milford, MA) coupled to the HPLC via a T-connector.
  • Spectra were acquired in the positive-ion mode on a Waters Ultima API mass spectrometer (Waters, Milford, MA) equipped with a Z-spray source.
  • the source temperature was 80°C
  • the cone and RF lens 1 potentials were 100 and 38 V, respectively.
  • the mass spectrometer was operated in single-reflector mode to achieve a resolution of 10 000 (full width half maximum definition).
  • the mass scale was calibrated using [Glu1]-fibrinopeptide B.
  • HDX-MS shows no difference in NT A72R between pH 7.0 and 6.0 ( Fig. 3 ). Deuterium incorporation for each peptic peptide was observed. HDX-MS of NT A72R did not show any significant changes between pH 7 and pH 6, while wildtype NT shows a reduced HDX, in particular in helix 3 spanning amino acid residues 63-84. The reduced HDX of wildtype NT indicates global stabilisation at low pH and is in line with the increased melting temperature of NT at low pH.
  • NMR experiments were performed at 298 K on a Varian Unity Inova 600 MHz spectrometer equipped with an HCN triple resonance PFG cold probe.
  • the NMR sample of wt NT sample contained 1.9 mM protein, 20 mM sodium phosphate (pH 7.2), 300 mM NaCl, 5% (v/v) D 2 O and 0.03% NaN 3 (w/v).
  • the 15 N-HSQC spectrum of NT A72R showed narrow and well-dispersed resonances even in the presence of low salt concentrations at pH 7.0 (data not shown), indicating that the protein is folded and monomeric at these conditions.
  • Nearly complete resonance assignments for the polypeptide backbone nuclei (HN, 15 N, 13 C ⁇ , 13 C' and 13 C ⁇ ) were obtained through analysis of triple-resonance 3D NMR spectra. Amino acid side chains were assigned using 3D 15 N-NOESY-HSQC and two 13 C-NOESY-HSQC spectra.
  • NT A72R is representative of the monomeric form of wt NT
  • the same procedure as for NT A72R was used to obtain resonance assignments and to determine the structure.
  • the resulting wt NT monomer structure is of equal quality to the NT A72R structure.
  • it is essentially identical with the NMR and X-ray structures of NT A72R , including a highly similar side chain arrangement near Trp10. This leads us to conclude that the NT A72R mutation successfully locks the protein in the wildtype-like monomeric form.
  • the crystal structure of NT A72R was solved to 1.7 ⁇ resolution by molecular replacement using the NMR structure as the search model.
  • the mutated NT is monomeric, with one monomer in the asymmetric unit.
  • the first six and the last seven residues of the polypeptide chain as well as two glycine residues in the loop between helices 3 and 4 are disordered and have not been modeled. These residues also show increased mobility and disorder in solution as manifested by 15 N ⁇ 1H ⁇ -NOE values below 0.6 and higher local RMSD values.
  • Fig. 4 Superpositioning in Fig. 4 of the monomeric (black) and dimeric (white/grey) NT subunit structures reveals that the helices of the five-helix bundle are significantly shifted in the two forms ( Fig 4 ).
  • Helix 2 (residues 35-60) and 3 (residues 63-84) form one unit, while helix 1 (residues 10-30), 4 (residues 90-110) and 5 (residues 113-130) form a second, with the two units shifted approximately as rigid bodies with respect to each other.
  • the subunit interface in dimeric NT is formed by helices 2, 3, and 5. Since helix 2 and 3 form a more or less rigid unit, two wt subunits in the monomeric conformation might potentially be able to dimerize via these two helices. However, modeling shows that this would require significant movement of helix 5 to resolve clashes.
  • dimeric NT the two highly conserved residues D40 at the beginning of helix 2 and E84 at the end of helix 3 form an intramolecular handshake interaction. This interaction is lost in monomeric NT and NT A72R , where the two carboxylates are more than 10 ⁇ apart in monomeric NT and NT A72R . This is accompanied by unwinding of the last half-turn of helix 3 in monomeric NT. Previous results indicate that NT can interconvert between different conformations and quaternary states.
  • the x-ray structure of wt NT shows a dimer in which a Asp40-Glu84 handshake interaction, intersubunit charge interactions, as well as the C-terminal region show conformational polymorphisms.
  • NT can exist as a monomer, be locked in a monomer form, and defines the conformational changes required for conversion to the dimer observed by x-ray crystallography.
  • the folds of monomeric and dimeric NT domains are very similar and the locations of the ⁇ -helices in the polypeptide chains are virtually identical, but distinct changes in local conformations are seen.
  • Relocation of the single tryptophan of NT, Trp10, from a buried to an exposed site is a main difference and is readily monitored by fluorescence spectroscopy ( Fig. 2 ). Trp10 is present in most of the sequenced species variants of NT, but can be replaced with Phe in some species.
  • Expression vectors were constructed to produce SP-C33Leu, miniBLeu and proSP-C(86-197), respectively, as C-terminal fusions to HisNT A72R (SEQ ID NOS 7, 9 and 11, respectively).
  • the corresponding fusion proteins HisNT A72R MetSP-C33Leu (SEQ ID NO 8), HisNT A72R MetMiniBLeu (SEQ ID NO 10) and HisNT A72R proSP-C(86-197) SEQ ID NO 12
  • SEQ ID NO 12 HisNT A72R proSP-C(86-197)
  • the yields obtained were: HisNT A72R MetSP-C33Leu above 300 mg/L culture of purified fusion protein, and above 50 mg/L of purified target protein HisNT A72R MetMiniBLeu above 50 mg/L culture of fusion protein HisNT A72R proSP-C(86-197) above 0.5 mg/L culture of fusion protein
  • Expression vectors are constructed to produce a number of desirable proteins and polypeptides as a C-terminal fusion to His-protease 3C cleavage site-NT A72R - TEV protease cleavage site.
  • the corresponding fusion proteins (SEQ ID NO 13-34 and corresponding sequences for fusion proteins incorporating the desirable proteins/polypeptide 4repCT, LL37/hCAP18, SPC-33, Bri2(76-231), Bri2-NL(90-236), Bri2(113-266), Bri2(125-231), Bri2(232-236), and Bri2-hairpin(244-266)) are expressed, and the desirable proteins and polypeptides are released and purified essentially as set out in Example 1.
  • Desirable proteins Construct ID SGC Protein Family ACOT9A-c009 Lipid signalling-Other ALKA-c023 Kinase-TK APOBEC3AA-c002 Cytosine deaminases BIRC1A-c050 Apoptosis-Inflammation Domains CSADA-c006 aa metabolic enzymes DDX53A-c006 ATPases - DExD-DExH RNA helicases DDX53A-c013 ATPases - DExD-DExH RNA helicases DOCK1A-c021 Lipid signalling-Other GARTA-c015 Nucleotide metabolism-Others GARTA-c017 Nucleotide metabolism-Others GLE1A-c007 Miscellaneous GMPSA-c020 Nucleotide metabolism-Others ITM2BA-c006 BRICHOS ITPKCA-c208 Kinase-PIK MYO

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EP3168228A1 (fr) 2015-11-13 2017-05-17 Spiber Technologies AB Domaine n-terminal monomère de la protéine de soie d'araignée et ses utilisations
WO2017081239A1 (fr) * 2015-11-13 2017-05-18 Spiber Technologies Ab Domaine n-terminal à charge inversée de protéine de soie d'araignée et son utilisation
WO2017122969A1 (fr) * 2016-01-15 2017-07-20 (주)넥스젠바이오텍 Protéine de fusion d'hormone de croissance humaine présentant une stabilité thermique élevée et composition cosmétique la contenant comme principe actif pour améliorer les rides cutanées et maintenir l'élasticité
EP3263593A1 (fr) * 2016-07-01 2018-01-03 Anna Rising Protéines de soie d'araignée mises au point par génie génétique et leurs utilisations
US20190085053A1 (en) * 2015-12-17 2019-03-21 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Synthetic lung surfactant with enhanced stability and effectiveness
US11179446B2 (en) * 2016-03-07 2021-11-23 Lundquist Institute For Biomedical Innovation At Harbor—Ucla Medical Center Compositions and method for administering PPARgamma agonists, surfactant peptides and phosopholipids

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IL259287B (en) * 2015-11-13 2022-08-01 Spiber Tech Ab N-terminal region with charge reversal of spider silk protein
WO2017081239A1 (fr) * 2015-11-13 2017-05-18 Spiber Technologies Ab Domaine n-terminal à charge inversée de protéine de soie d'araignée et son utilisation
EP3168228A1 (fr) 2015-11-13 2017-05-17 Spiber Technologies AB Domaine n-terminal monomère de la protéine de soie d'araignée et ses utilisations
US11214601B2 (en) 2015-11-13 2022-01-04 Spiber Technologies Ab Charge-reversed N-terminal spider silk protein domain and uses thereof
CN108431027A (zh) * 2015-11-13 2018-08-21 思百博技术股份公司 电荷逆转的n端蜘蛛丝蛋白结构域及其用途
JP2019503986A (ja) * 2015-11-13 2019-02-14 スピベル テクノロジーズ アクティエボラーグ 電荷反転ｎ−末端スパイダーシルクタンパク質ドメイン及びその使用
US10626152B2 (en) 2015-11-13 2020-04-21 Spiber Technologies Ab Charge-reversed N-terminal spider silk protein domain and uses thereof
US20190085053A1 (en) * 2015-12-17 2019-03-21 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center Synthetic lung surfactant with enhanced stability and effectiveness
US10717777B2 (en) * 2015-12-17 2020-07-21 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center Synthetic lung surfactant with enhanced stability and effectiveness
US20230107377A1 (en) * 2015-12-17 2023-04-06 Lundquist Institute For Biomedical Innovation At Harbor-Ucla Medical Center Synthetic lung surfactant with enhanced stability and effectiveness
WO2017122969A1 (fr) * 2016-01-15 2017-07-20 (주)넥스젠바이오텍 Protéine de fusion d'hormone de croissance humaine présentant une stabilité thermique élevée et composition cosmétique la contenant comme principe actif pour améliorer les rides cutanées et maintenir l'élasticité
US11166898B2 (en) 2016-01-15 2021-11-09 Sun Kyo LEE Human growth hormone fusion protein with enhanced thermal stability and cosmetic composition for anti-wrinkle and maintaining skin elasticity comprising the same as effective component
US11179446B2 (en) * 2016-03-07 2021-11-23 Lundquist Institute For Biomedical Innovation At Harbor—Ucla Medical Center Compositions and method for administering PPARgamma agonists, surfactant peptides and phosopholipids
US11951158B2 (en) 2016-03-07 2024-04-09 Lindquist Institute For Biomedical Innovation At Harbor-Ucla Medical Center Compositions and methods for administering PPARγ agonists, surfactant peptides and phospholipids
EP3263593A1 (fr) * 2016-07-01 2018-01-03 Anna Rising Protéines de soie d'araignée mises au point par génie génétique et leurs utilisations
JP2020536843A (ja) * 2016-07-01 2020-12-17 スパイバー テクノロジーズ アクティエボラーグ 改変クモ糸タンパク質及びその用途
CN109689680B (zh) * 2016-07-01 2022-09-16 思百博技术股份公司 工程化蜘蛛丝蛋白及其用途
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